Channeling (preferential use of intermediates bound to enzyme complexes) has been proposed in several metabolic pathways. In systems where it operates, it could increase the overall catalytic efficiency. No evidence could be found for channeling of substrates in the biosynthetic pathway of histidine. Radioactive imidazole glycerol phosphate (IGP) was synthesized chemically and its composition verified by a number of criteria. An electrophoretic assay for separating each of the intermediates in histidine biosynthesis from IGP to histidine was developed. Extracts were prepared from bacteria containing high levels of the enzymes and the kinetics of disappearance of IGP and the appearance of the intermediate and products were carefully monitored. Even in the presence of polyethylene glycol to provide crowded conditions, no channeling of IGP to histidine was observed.